Steve Ealick's Research Group
Thiamin Phosphate Synthase from Bacillus subtilis
2TPS (complexed with thiamin phosphate and pyrophosphate 2-)
*1G4E (S130A mutant)
*1G4P (S130A mutant, complexed with 4-amino-2-trifluoromethyl-5-hydroxymethylpyrimidine pyrophosphate)
*1G4S (S130A mutant, complexed with thiamin phosphate )
*1G4T (S130A mutant, complexed with trifluoro-thiamin phosphate)
*1G67 (S130A mutant, complexed with 2-methyl-5-methylene-5H-pyrimidin-4-ylideneamine, 4-methyl-5-hydroxyethylthiazole phosphate and pyrophosphate)
*1G69 (S130A mutant, complexed with 2-methyl-5-methylene-5H-pyrimidin-4-ylideneamine, 4-methyl-5-hydroxyethylthiazole phosphate and pyrophosphate-fast soak)
*1G6C (S130A mutant, complexed with 2-trifluoromethyl-5-methylene-5H-pyrimidin-4-ylideneamine, 4-methyl-5-hydroxyethylthiazole phosphate and pyrophosphate)
#3O15 with carboxylated thiazole phosphate
#3O16 K159A mutant
The biosynthesis of thiamin, which is an essential nutrient for humans, involves the separate formation of 4-amino-2-methyl-5-hydroxymethylpyrimidine phyrophosphate (HMP-PP) and 4-methyl-5-hydroxyethylthiazole phosphate (Thz-P). These are coupled to form thiamin phosphate (TP) in a reaction catalyzed by thiamin phosphate synthase. For this work, thiamin phosphate synthase was overexpressed and purified from Bacillus subtilis. The X-ray structure of the small (25,277 Da) enzyme, complexed with the product thiamin phosphate and pyrophosphate, was determined at 1.25 Å.
Thiamin phosphate synthase is an α/β protein with a (βα)8 barrel fold. The enzymatic products, thiamin phosphate, and pyrophosphate are shown looking down the barrel.
Click the image to enlarge.
The pyrophosphate is stabilized by extensive electrostatic and hydrogen bonding interactions, especially the one between S130 and pyrophosphate, as shown in this schematic representation of the active site. Thiamine phosphate binds in a "V" conformation, which is maintained predominantly by van der Waals interactions between Ile186 and the pyrimidine and thiazolium rings. Interactions between thiamine phosphate and active site residues are shown in this schematic representation.
Chiu H-J., Reddick JJ, Begley TP, and Ealick SE. Crystal Structure of Thiamin Phosphate Synthase from Bacillus subtilis at 1.25 Å Resolution. Biochemistry 38:6460-6470 (1999).
*Peapus DH, Chiu H-J, Campobasso N, Reddick JJ, Begley TP, and Ealick SE. Structural Characterization of the Enzyme-Substrate, Enzyme-Intermediate, and Enzyme-Product Complexes of Thiamin Phosphate Synthase. Biochemistry 40:10103-10114 (2001).